Total RNA from the frozen samples was extracted using the TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocol. Genomic DNA contamination was first removed using RNA-free DNase I, and the RNA integrity and quality were then analyzed using a Bioanalyzer (Agilent, USA). The RNA integrity threshold was RIN ? 6.8. PolyA(+) RNA-seq libraries were constructed using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations. The resulting cDNA was first cleaved into 300-500-bp fragments to construct libraries according to the manufacturer’s instructions, and the libraries were then sequenced using the Illumina HiSeq 2000/2500 platform